The long term objective of this grant proposal is to elucidate the function of RAS proteins. The RAS gene family has been implicated in the control of normal cell proliferation and differentiation, and in the development of various human cancers. The high degree of evolutionary conservation among RAS genes is exemplified by the structural and functional homology between yeast and mammalian RAS proteins. The goal of the proposed studies is to identify and characterize the proteins that constitute the RAS signalling pathway in yeast. For these purposes the studies utilize dominant interfering RAS mutants that block the transmission of RAS signals in yeast and in higher eukaryotes. These mutants provide a novel and powerful probe to examine RAS function. Two complementing experimental approaches will be employed in these studies. One approach is to identify and characterize the genes that can restore transmission of RAS signals in the presence of these interfering mutants. The second approach will involve the establishment of a direct biochemical assay to detect the physical interactions of these interfering RAS mutants with cellular proteins. These studies will be extended to higher eukaryotes by examining the effects yeast proteins that interact directly with RAS have on the function of RAS in amphibian oocytes and in cultured mammalian cells. Additionally, if time permits, mammalian homologs of yeast proteins that interact directly with RAS will be isolated. The identification of the proteins that mediate RAS function, and of proteins that associate directly with RAS will constitute an important contribution to the understanding of RAS signal transduction pathways in yeast and in higher eukaryotes.